The invention relates to the use of genetically modified earliest haematopoietic and mesenchymal stem cells (which are negative for the expression of the CD34 surface molecule) in individual gene therapy of mono- or oligogenetic diseases, respectively, diseases in blood formation as well as chronic disorders. For this purpose, endogenous CD34-negative adherently growing stem cell cultures are established from peripheral blood of the patient and efficiently transfected or infected, respectively, with gene constructs. On a long term basis, the gene products of said genes should substitute defective or missing proteins or factors in the patient organism, respectively, or enable a cell therapy.
The aim of somatic gene therapy or cell therapy, respectively, is the effective transfer of genetic material into the organism. In somatic gene therapy, genetic defects in body cells are corrected or genes encoding therapeutically useful gene products are introduced into cells. The gene-therapeutic alteration is not transmitted to the offspring. The introduction of genetic material into target cells may be done ex vivo as well as in vivo. Ex vivo means that the target cells are cultured outside of the body and are then reintroduced into the patient after introduction of the genetic material. However, the efficiency of somatic gene therapy is affected by the limited life of the transfected cells. Thus, cells having a particularly long life span are particularly suitable as the target cells for somatic gene therapy, such as haematopoietic stem cells. Heretofore, haeomatopoietic stem cells for transplantation purposes were obtained either from bone marrow of the donor or after enrichment steps from peripheral blood. The isolation of cells from the body of the patient or a near relative is referred to as allogenic bone marrow transplantation. Then, the frequently unselected mixture of stem cells and other bone marrow cells is reintroduced into the patient. Finally, the haematopoietic stem cells contained in said mixture migrate from the blood into the blood forming bone marrow to produce all cells of the blood forming system in a necessary amount. This type of transplantation is often associated with severe complications. Even the smallest tissue differences between donor and acceptor may result in highly dangerous complications for the patient. This risk must be weighed up against the actual disease, e.g. leukaemia. The donor-acceptor incompatibilities (graft versus host disease) are usually caused by cells contaminating the actual stem cell preparation. In particular, these are cells of the donor immune system.
To exclude this contamination of the bone marrow or stem cell transplant, respectively, methods have been developed to enrich the population of the haematopoietic stem cell. This cell which is also called pluripotent haematopoietic stem cell has been defined by the expression or non-expression of particular surface molecules so far. This pluripotent haematopoietic stem cell is able to produce the human haematopoietic cell linesxe2x80x94for example B cells, T cells, leukocytes, platelets or erythrocytesxe2x80x94via additional precursor cells. At the moment, the determination of a haematopoietic cell as a pluripotent haematopoietic stem cell is defined by the expression of the so-called CD34 molecule and simultaneously by the non-expression of other surface molecules, such as CD5. The CD34 molecule is a strongly negatively charged proteoglycane of the mucine family with a molecular weight of about 105 to 120 kD. Cellpro Inc. company, Seattle, USA, has developed a method for the purification of CD34 positive cells by means of an affinity chromatography (U.S. Pat. Nos. 5,215,927, 5,262,334, 5,240,856, 5,225 353, EP 526,577 B and EP 260,280 B). Simultaneously, CD34-negative cells form the pool for mesenchymal stem cells.
At the moment, for somatic gene therapy by means of haematopoietic stem cells CD34-positive cells are isolated from peripheral blood of the patient after stimulation of said cells with growth factors, e.g. G-CSF (Neupogen-R), and are reintroduced after genetic manipulation into the patient to reconstitute the lethally irradiated and chemotherapeutically treated bone marrow. Up to now, haematopoietic stem cells modified in this manner have been employed in particular for specific immune deficiency syndromes, such as adenosine desaminase defect, SCID syndrome or HIV infection, for metabolic diseases, such as Morbus Gaucher, in disorders of the blood formation, e.g. specific forms of thalassaemia and in malignant diseases, such as leukaemia.
Due to ethical and social reasons, this method is however restricted to the autologous or blood stem cell donation by near relatives, since for the accumulation of the stem cells in peripheral blood a growth factor has to be administered to the donor or patient, respectively. Up to now, it was not possible to predict the long term effect of said growth factor on a possible expansion of a leukaemia clone or a possible transformation of a healthy blood stem cell.
Furthermore, the somatic gene therapy by means of haematopoietic stem cells causes certain technical difficulties. Only a small portion of the haematopoietic stem cells transfected with therapeutic genes or gene constructs receives the genetic modification or no gene product will be produced. Thus, the efficiency of said method is very low at the moment; c.f. Huss, R. Infusionsthera. Transfusionsmed. 23 (1996) 147-160.
There is the need to provide improved means and methods which may be used in gene therapy.
The object is solved by the subject matters mentioned in the claims.
Thus, the subject matter of the invention are genetically modified CD34 negative adherently growing stem cells.
In a preferred embodiment the life span or the ability to divide, respectively, is prolonged by transient immortalization.